title: Sample 19
description: Protocols: The model used to investigate biofilm formation over time in S. Typhimurium was identical to that used in our investigations with E. coli (Holden et al., 2021). Parallel cultures of 5 mL LB broth (without salt) were inoculated with approximately 107 cells from the pooled S. Typhimurium mutant library. This was done with and without 1 mM IPTG to induce expression from the transposon-located promoter. Cultures were grown in 6-well plates containing 40 sterile 5 mm glass beads (Sigma) per well and incubated at 30 ˚C with light agitation. After 12-, 24-, and 48-hours post-inoculation, 2 mL of planktonic sample was collected from each culture and 70 beads were taken to constitute the biofilm sample. Beads were washed twice in sterile PBS and vortexed in tubes containing PBS to resuspend cells from the biofilm. Both planktonic and biofilm samples were centrifuged at 2100 x g to form pellets for DNA extraction. All conditions were run with two independent identical replicates. DNA was extracted from pellets following the protocol described by Trampari et al. (2021, npj Biofilms) customised sequencing libraries were prepared in the same way as described by Holden et al (2021, Microbial Genomics). DNA was fragmented using the MuSeek DNA fragment library preparation kit (ThermoFisher) and were amplified by PCR using customised sequencing primers specific for the transposon tag. Fragments between 300 and 500 bp were size selected using AMPure beads (Beckman Coulter).
attributes.tag: ENA first public
attributes.value: 2022-05-27
attributes.tag: ENA last update
attributes.value: 2022-05-27
attributes.tag: External Id
attributes.value: SAMEA14449711
attributes.tag: INSDC center alias
attributes.value: Quadram Institute Bioscience
attributes.tag: INSDC center name
attributes.value: Quadram Institute Bioscience
attributes.tag: INSDC first public
attributes.value: 2022-05-27T00:15:35Z
attributes.tag: INSDC last update
attributes.value: 2022-05-27T00:15:35Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11765:Sample 19
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: growth condition
attributes.value: biofilm
attributes.tag: replicate
attributes.value: 1
attributes.tag: sample name
attributes.value: E-MTAB-11765:Sample 19
attributes.tag: stimulus
attributes.value: promoter induction with IPTG
attributes.tag: serovar
attributes.value: Typhimurium
attributes.tag: sub_species
attributes.value: enterica